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The role of the cell envelope in cell division has been examined using mutants of Escherichia coli which are temperature-sensitive in the process of cell division. These mutants grow normally at 30 C but exhibit morphological changes at 42 C. Two assays for peptidoglycan-specific autolytic enzymes have been developed. One depends on the prevention of synthesis of autolytic enzymes using chloramphenicol, while the autolytic capacity is determined by observing the rate of cell lysis in the presence of low levels of ampicillin. The second assay is based on the in vitro release of specific radioactive fragments from peptidoglycan previously labelled with radioactive diaminopimelic acid. The majority of cells in an exponential population of E. coli B/r/l are not lysed by penicillin if chloramphenicol is added. However, larger cells are particularly susceptible to lysis. Three types of lysis have been defined by observing rates of lysis of synchronous cultures at different ages and at different growth rates: (A) lysis associated with cross-wall formation during cell division; (B) lysis associated with initiation and segregation of the replication of DNA; and (C) lysis associated with general expansion of the cell wall. Filamentous mutants of E. coli which segregate DNA exhibit lysis in excess of type C while filaments formed by inhibition of DNA synthesis exhibit lysis only of type C. The autolytic enzymes of E. coli appear to be tightly bound to localized areas of the cell envelope as determined by the in vitro assay. Qualitative differences between the autolytic activities of normal cells and of filaments formed by inhibition of DNA synthesis are described. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

A temperature-sensitive division mutant, Escherichia coli BUG-6, has been investigated. This organism divides normally when grown at 30 C but fails to divide at 42 C. Growth continues at 42 C to produce very long, multinucleate filamentous cells. When returned to 30 C, or a high osmotic environment is added at 42 C, the filamentous cells divide rapidly to produce cells of normal size. The kinetics of cell division of the filaments at 30 C depends on the period at 42 C. During filament formation, DNA, RNA and protein synthesis continue as measured by radio-isotopic incorporation and chemical cell fractionation. DNA segregation occurs as shown by autoradiography. The rapid division of filaments replaced at 30 C cannot be prevented by novobiocin or cycloserine but is prevented by vancomycin and penicillin suggesting that de novo synthesis of cell wall precursors is not required for division but that positioning and cross-linking of the precursors is required. Filaments growing at 42 C were treated with nalidixic acid for different lengths of time. On returning these filaments to 30 C in nalidixic acid the number of divisions was proportional to the length of time at 42 C in the absence of nalidixic acid, ie. proportional to the amount of DNA synthesized at 42 C. Inhibition of protein synthesis, by chloramphenicol, does not prevent the division of filaments on replacing at 30 C provided that the period of filamentation at 42 C was greater than 6 minutes and less than 110 minutes. The maximum amount of division in the absence of protein synthesis occurred after a longer lag and slower than in non-inhibited control cultures. If protein synthesis was inhibited in filaments at 42 C the ability of such treated cells to divide at 30 C was rapidly lost. This loss of 'division potential’ has a half-life of about 0.5 minutes, ie. 0.5 minutes of protein inhibition at 42 C reduces the subsequent division at 30 C by 50%. The normal presence of 'division potential', therefore requires the synthetic doubling rate to be in excess of 0.5 minutes. Very short periods at 42 C indicate that 10 minutes incubation at 42 C is required to produce this extremely fast synthetic rate. A model for the production and expression of 'division potential' is presented. A biochemical analysis of the cell envelope of the filamentous cells and of normally dividing cells is presented. The major phospholipid compositions are the same. However, the fatty acid contents differ especially with regard to the cyclic fatty acids. When the filaments are allowed to divide by replacing at 30 C their fatty acid composition very rapidly reverts to that of normally dividing cells. The rates of individual phospholipid syntheses appear to change during the rapid cell division phase, however this may be an artifact resulting from an overall increase in the rate of phospholipid synthesis during this period. An analysis of the proteins within the cell envelope by radio-active double labelling techniques and followed by gel electrophoresis indicates that a protein(s) of molecular weight 80,000 - 90,000 exists in the envelope of filamentous cells which is not in the envelope of normal cells or is made at a much slower rate in normal cells. The protein is not incorporated into septa during the division of filaments at 30 C and little turnover occurs in the major proteins synthesized at 42 C when these cells are placed at 30 C. The possibility exists, however, that this protein is a product of filamentation and not the temperature sensitive gene product. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Temperature-sensitive mutants have been used in this study to examine development in Paramecium tetraurelia. Ten mutations affecting development were described. Since two of the mutants were allelic, the effects of nine genes on Paramecium development were studied. Of these, two affect formation of the fission-zone (called dfz or defective fission-zone mutants), five affect constriction of the fission-furrow (called dc or defective constriction mutants), and two produce a reduction in cell size (called sm or small mutants). Morphometric measurements were made on inter-fission and dividing wild-type and mutant cells to examine two aspects of Paramecium development: changes in cell shape and size which preceed and accompany cell division and positioning of new structures on the cell surface during cell division. This analysis suggested the following hypotheses: 1. The shape and size changes which preceed and accompany cell division in Paramecium are causally related to cell division. Defective constriction mutants undergo exaggerated contraction prior to fission-furrow formation. This contraction appears to interfere with the decrease in cell width which ordinarily occurs during division. Although the mutant cells are able to make a normal amount of furrow surface, the abnormal cell width prevents furrow completion. Premature contractions seen in dfz mutants similarly interfere with furrow formation. 2. Surface growth in Paramecium is dependent on prior basal body proliferation. Basal bodies appear to act as organizing centres for surface growth. Reduced basal body proliferation in mutant cells was always associated with reduced surface growth: There was a consistent relationship between the number of new basal bodies produced proceeding cell division and the amount of surface growth which occurred. The order of the causal relationship was suggested by the observation that basal body proliferation was completed prior to the beginning of surface growth. 3. The positioning of new structures during cell division in Paramecium can be affected by the pre-existing cell shape, size, or structure. This model, called mechanical guidance, was based on observations of the movement and positioning of the vestibule (the opening leading to the mouth) in wild-type and mutant cells. This model was discussed in relation to other developmental mechanisms proposed to account for protozoan morphogenesis. / Science, Faculty of / Zoology, Department of / Graduate

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In this study, we investigated the effects of the peripheral benzodiazepine ligands, Ro5-4864 (putative antagonist) and PK 11195 (putative antagonist) on prolactin stimulated mitogenesis in Nb2 cells. Ro5-4864 and PK 11195 at 10⁻⁹ M maximally enhanced prolactin stimulated mitogenesis. At 10⁻⁶ M Ro5-4864 inhibited prolactin stimulated mitogenesis. Clonazepam, a ligand for the central benzodiazepine receptor had no effect on mitogenesis. Interaction studies were undertaken to determine if Ro5-4864 and PK 11195 act on the same site. The ability of each ligand to enhance the mitogenic action of prolactin was blocked by a 10⁻⁶ M concentration of the other ligand. Finally, simultaneous addition of 10⁻⁹ M of the ligands resulted in no additive effect over each ligand alone. These data show that peripheral benzodiazepine ligands modulate prolactin-stimulated mitogenesis and suggests they interact at the same binding site.

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